In general, slides are used for microscopy. For instance, cell or tissue specimens are applied to or positioned on a slide. The slide with the cell or tissue specimen is then placed on a microscope stage of a microscope so that the cell or tissue specimen may be examined with the microscope.
In addition, microscope scanning systems are used for automatically finding objects like cells in a tissue specimen that is positioned on a slide. These may be, for instance, cells with a certain property (e.g. a marking) that are present in a tissue slice that is on the slide. A marking may be for instance staining of a cell.
Microscope stages and scanning stages are equipped as measuring elements with coordinate systems so that, for instance, the position of the found object may be precisely determined using coordinates from the measuring element and/or slide.
As a rule the position of the found objects is stored. This may be done so that after a specimen (e.g. tissue specimen) on a slide has been scanned, the found objects (e.g. cells) can be automatically relocalized or reexamined, i.e. re-presented to the viewer for visual examination. An absolute X-Y-Z coordinate system is not necessary to guarantee precise relocalization as long as the relocalization takes place immediately after the specimen has been scanned, i.e. the specimen has not been removed from its original position on the scanning stage. In this case it is enough to approach the relative coordinates stored during the search.
In many cases it is not possible to relocalize immediately after the specimen has been scanned. This is especially the case when the scan is performed in “batch” mode. In this case, for instance, a certain number of specimens on the slides are automatically scanned first, e.g. overnight, and the found objects are not to be reviewed until the next morning.
In such a mode, it will regularly be necessary to remove the scanned slide from the stage using an automatic exchanger/robot module and to load a new set of slides. This is preferably not performed with individual slides, since direct manipulation of glass slides is prone to faults due to the normal tolerances of the slide dimensions. Instead, slides are placed into a frame (hereinafter referred to as a “slide frame”), which is then exchanged as a unit. Depending on size and configuration, one slide frame may accommodate one or a plurality of slides.
For receiving slides in a slide frame, corresponding slide seats are provided so that one slide may be placed into each slide seat.
The slide frames are stored for instance in a magazine, from which magazine the robot module can remove the slide frames. Once the slide(s) that is/are arranged in the slide frame has/have been scanned, the slide frame may be returned to the magazine.
In addition, specimen exchangers may be provided that have for instance 1 to 11 magazines, each magazine accommodating e.g. 16 slide frames, each with 5 slides. In order to be able to receive the slide or plurality of slides in one slide frame, the slide frames have one slide frame seat for each slide.
A 3-point self-centering support provides positioning accuracy for the slide frames themselves on the scanning stage.
The slide frames are not exactly identical; on the contrary, there are production tolerances. Although as a rule these production tolerances lead to positioning errors of a few tenths of a millimeter, this is still enough for instance to displace a cell out of the microscope image field when this cell is relocalized based on relative coordinates.
Consequently, the “absolute” coordinates for any position on a slide must be known for a slide frame in which the slide is arranged in order to be able to reliably relocalize any coordinates for an object that is mounted on the slide, regardless of the slide frame used and the slide frame receiving unit used.
In practice, it may happen that one slide is placed into successive different slide frames. For instance, one slide may contain a tissue specimen that was stained in order to mark a certain cell(s) with color. The slide is now placed into a “first” slide frame in order to scan the slide automatically. The stained cells are detected and their position on the slide is determined and stored. After this automatic scanning has concluded, the slide is removed from the “first” slide frame in order to destain the slide. Then the cells are stained again in order to obtain additional information about the cells to be examined. To this end, the slide is placed into a “second” slide frame and is again placed under the microscope for examination. It is necessary to be able to perform precise relocalization in order to be able to, immediately and in a simple manner, further examine the cells that were previously examined.
Therefore, for precise relocalization the position of a slide must be calibrated in each slide frame so that a slide may be placed into different slide frames and the positioning coordinates for a “found” object are accurate regardless of the slide frame used.
The same is true when a slide frame includes a plurality of slide seats. In this case, a calibration must be conducted for each slide seat so that the same slide may be placed into any slide seat of the slide frame and a “found” object can still be reliably relocalized.
In the past, calibration of a slide frame or a slide seat was performed such that a special slide (calibration slide) that was provided with a highly precise coordinate grid was placed into the slide seat that was to be calibrated. Then three defined coordinates (three calibration positions) that were covered with cross-hairs were approached in succession, and this was displayed in a live image on a monitor for the microscope scanning system. Software stored the coordinates for the scanning stage that belonged to these three calibration positions and the coordinates were used for converting coordinates between different positions on the slide with respect to the slide frame.
However, the current generation of specimen exchanger can include up to 880 slides, so that this approach to calibrating slide frames is no longer practical.
This is because it is necessary to perform a placement process for the calibration slide for each slide seat for a slide in a slide frame and it is also necessary to perform three precise manual positioning operations. Interactive centering of the three grid positions is time-consuming, as well. If these steps are to be skipped, a large number of identical calibration slides (for instance, 80 calibration slides) are needed to be able to automatically calibrate a magazine of slide frames. The costs for the high-precision calibration slides are a drawback in this process in accordance with the prior art, and the calibration precision is limited by the precision of the calibration slide. Producing calibration slides with tolerances of 1 to 5 μm is complex, and therefore expensive.